Herbo-mineral formulation for prevention, treatment and management of renal disorders and method of preparation thereof

ABSTRACT

Herbo-mineral formulation for prevention and treatment of Renal disorders are disclosed herein. The formulation includes combination of herb and mineral elements for treating Renal disorders. Renal disorders include any condition associated with Kidney function such as Chronic renal failure.

CROSS REFERENCE TO RELATED APPLICATION

This application is based on and derives the benefit of U.S. ProvisionalApplication 62/531,216 filed on Jul. 11, 2017, the contents of which areincorporated herein by reference.

TECHNICAL FIELD

The embodiments disclosed in this specification relates to herbo-mineralformulations effective in the treatment, management and prevention ofRenal Disorders, and more particularly as formulations that act asnephron-protective agents. It also relates to the process of preparationof such formulations.

BACKGROUND

Renal Disorders are diseases related to the Kidney function. Kidneys'ability to cleanse blood, maintain water and salt balance, and regulatevolume and composition of bodily fluids, makes it one of the mostimportant organs in the human body. Any impairment in this ability ofthe kidney could have grave consequences. Therefore, maintainingwell-functioning kidneys is crucial for good health.

Renal disorders may be due to many reasons as in the case of AcuteKidney Injury (AKI) wherein kidney malfunction occurs due to blood loss,dehydration or use of medicines, etc. AKI is usually a more sudden andreversible condition. On the other hand, Chronic Renal Failure (CRF) isan irreversible condition with progressive loss in kidney function. Oneof the primary causes of CRF include Diabetes causing diabeticnephropathy. Others primary indications for kidney dysfunction includehypertension, glomerulonephritis, infection and so on.

Various methods of treating renal disorders are known. The appropriateline of treatment may be chosen based on the cause of Renal Disorder. Insome cases, medication may be directed towards treating primarycomplications affecting the kidney function. Such medication may includemedication for treating diabetes mellitus, high blood pressure, highcholesterol, anemia, etc. Further, in cases of end stage kidney disease,treatment would include dialysis and kidney transplant. Although, thesymptomatic treatment regime, often implemented in the practice ofallopathic medicine may help in alleviating the symptoms temporarily,they often do not focus on improving the overall health of kidneys.

The core philosophy of Ayurvedic medicine on the other hand is toattain/maintain harmony in the system and hence its approach works onimproving the health of the body rather than treating symptoms.Ayurvedic interventions have also been known in treating RenalDisorders. Many herbal formulations have been developed based on theknowledge of the healing properties of various herbs. Formulationshaving herbs such as Tinospora cordifolia, Terminalia chebula, Boerhaviadiffusa, etc have been developed. However, the effectiveness of suchformulations is arguable. There exists a need for an effective method oftreating/managing Renal Disorders.

OBJECTS OF THE DISCLOSED EMBODIMENTS

The principal object of the embodiments disclosed herein is to provide acomposition and method of treating Renal disorders.

A second object of the embodiments disclosed herein is to provide acomposition and method of managing Renal disorders.

Another object of the embodiments disclosed herein is to provide acomposition and method of preventing Renal disorders.

Another object of the embodiments disclosed herein is to provide aherbo-mineral formulation and a method for its preparation.

These and other objects of the embodiments herein will be betterappreciated and understood when considered in conjunction with thefollowing description and the accompanying drawings. It should beunderstood, however, that the following descriptions, while indicatingpreferred embodiments and numerous specific details thereof, are givenby way of illustration and not of limitation. Many changes andmodifications may be made within the scope of the embodiments hereinwithout departing from the spirit thereof, and the embodiments hereininclude all such modifications.

BRIEF DESCRIPTION OF FIGURES

The embodiments disclosed herein are illustrated in the accompanyingdrawings, throughout which like reference letters indicate correspondingparts in the various figures. The embodiments herein will be betterunderstood from the following description with reference to thedrawings, in which:

FIG. 1(a) depicts a flowchart for the preparation of Swarna MakshikaBhasma

FIG. 1(b) depicts a flowchart for the preparation of Mandura Bhasma;

FIG. 1(c) depicts a flowchart for the preparation of Loha Bhasma;

FIG. 2 depicts a flowchart for the preparation of fortified tablets;

FIG. 3 represents the histopathological observations of Kidneys of GroupI (Normal control) and Group II (Positive control);

FIG. 4 represents the histopathological observations of Kidneys of GroupIII and Group IV treated with the embodiments disclosed herein; and

FIG. 5 represents the histopathological observation of Liver of Group I,Group II, Group III and Group IV, according to the embodiments herein

DETAILED DESCRIPTION

The embodiments herein and the various features and advantageous detailsthereof are explained more fully with reference to the non-limitingembodiments that are illustrated in the accompanying drawings anddetailed in the following description. Descriptions of well-knowncomponents and processing techniques are omitted so as to notunnecessarily obscure the embodiments herein. The examples used hereinare intended merely to facilitate an understanding of ways in which theembodiments herein may be practiced and to further enable those of skillin the art to practice the embodiments herein. Accordingly, the examplesshould not be construed as limiting the scope of the embodiments herein.

The embodiments herein achieve a herbo-mineral formulation oftherapeutic value, and a process for the preparation of the formulation.The herbo-mineral formulation disclosed herein is useful in thetreatment, prevention and management of Renal Disorders. In variousembodiments herein, Renal Disorders may include any condition associatedwith Kidneys such as Nephritis, Nephrosis, Chronic kidney failure, etc.It has also been observed that the embodiments of the disclosedformulation may be instrumental in preventing the complications ofkidney diseases such as anemia, electrolyte imbalance, high bloodpressure, proteinuria, hyperkalemia, etc.

Further, the disclosed formulation also finds use in enhancingresistance of renal parenchyma against various types of nephrotoxicity.The various embodiments of the formulation also include a supplement foruse as a nephro-protective agent. The formulation may be used asmonotherapy or as an adjunct with other medication used to treat kidneydiseases. Accordingly, the embodiments disclosed herein achieve a methodfor the treating/managing/preventing renal disorders.

Formulation

The disclosed embodiments herein provide herbo-mineral formulationhaving a combination of selected herbs and minerals. In an embodiment,the herbo-mineral formulation includes herb element and mineral element.In another embodiment, the herbo-mineral formulation includes herbelement, mineral element and atleast one alkali. In yet anotherembodiment, the herbal formulation includes herb element, mineralelement, atleast one alkali and atleast one salt. In an embodiment, theherbo-mineral formulation may further include one or more suitableexcipient.

Herb Element

In an embodiment, the herb element includes the herbs selected from agroup consisting of Cinnamomum camphora, Acorus calamus, Saussurealappa, Andrographis paniculata, Tinospora cordifolia, Cedrus deodara,Curcuma longa, Aconitum heterophyllum, Berberis aristata, Piper longum,Plumbago rosea, Coriandrum sativum, Emblica officinalis, Terminaliachebula, Terminalia bellerica, Embelia ribes, Piper chaba, Piper longum,Saccharum officinarum, Commiphora mukul, Piper nigrum and Zingiberofficinalis or their extracts, or the active ingredients extracted fromthese herbs. In another embodiment, the herb element further includes atleast one of the herbs selected from Operculina terpethum, Baliospermummontanum, Cinnamomum tamala, Cinnamomum zeylanica, Elettaria cardamomum,Bamboo manna, Boerhavia diffusa, Cyperus rotundas, Bauhinia variegateand Holarrhena antidysenterica, or their extracts, or the activeingredients extracted from these herbs.

The herb element may include a specific part of the herb (also referredas herb component) such as roots, flowers, fruits, stem, bark, resin,rhizome, whole plant, extract etc. In an embodiment, the herb elementmay include roots of herbs selected from a group consisting of Acoruscalamus, Saussurea lappa, Aconitum heterophyllum, Berberis aristata,Piper longum, Piper chaba, Plumbago rosea, Operculina turpethum,Baliospermum montanum Elettaria cardamomum, Boerhavia diffusa andCyperus rotundas; fruits of herbs selected from a group consisting ofCoriandrum sativum, Emblica officinalis, Terminalia chebula, Terminaliabellerica, Piper longum, Embelia ribes, and Piper nigrum; crystals ofCinnamomum camphora; heartwood of Cedrus deodara; leaves of Cinnamomumtamala; whole plant of Andrographis paniculata; rhizome of Zingiberofficinalis and Curcuma longa; stem of Tinospora cordifolia Saccharumofficinarum and Piper chaba; and stem bark of Cinnamomum zeylanica,Bauhinia variegata and Holarrhena antidysenterica; or their extract.

The herb element may also include any form of secretion, resin ordischarge that may be exuded by the herb or any part of the herb. In anembodiment, the herb element may include secretion of Bamboo manna andoleo-gum resin of Commiphora mukul. However, it is also within the scopeof the claims provided herein for the herbo-mineral formulation toinclude other herb components such as leaf, flowers, etc. withoutotherwise deterring intended function of the herbo-mineral formulation.

The herb component maybe included in the formulation in any form that isgenerally known in the field. For example, the herb component may bedried, powdered, processed to form concentrates, extracts, etc. In onepreferred embodiment, the herb components are dried and powdered whichis further incorporated into the formulation. In another embodiment, theherb component includes stem (for example: stem of Saccharumofficinarum) which may be crushed to yield an extract which is furtherused in the formulation.

In an embodiment, the herb element includes the herbs Cinnamomumcamphora, Acorus calamus, Saussurea lappa, Andrographis paniculata,Tinospora cordifolia, Cedrus deodara, Curcuma longa, Aconitumheterophyllum, Berberis aristata, Piper longum, Plumbago rosea,Coriandrum sativum, Emblica officinalis, Terminalia chebula, Terminaliabellerica, Piper chaba, Embelia ribes, Piper longum, Piper nigrum andZingiber officinalis in an amount in the range of 1 to 4 wt %;Commiphora mukul in an amount in the range of 6 to 10 wt %; andSaccharum officinarum in an amount in the range of 2 to 6 wt %. Further,in another embodiment, the herb element includes at least one ofOperculina turpethum, Baliospermum montanum, Cinnamomum tamala,Cinnamomum zeylanica, Elettaria cardamomum, Bamboo manna, Boerhaviadiffusa, Cyperus rotundas, Bauhinia variegata and Holarrhenaantidysenterica in an amount in the range of ≤2 wt %.

Mineral Element

In an embodiment, the mineral element includes Bhasmas or calcinedpreparations such as Swarna Makshika bhasma, Mandura bhasma and Lohabhasma. Alternatively, the mineral element may also be selected from agroup consisting of at least one of iron rust, iron and copper pyrite.In the disclosed embodiments, the bhasmas along with the herb elementform bioavailable herbo-mineral complexes which are useful in treating,preventing and managing Kidney related complications. In an embodiment,the mineral element further includes Shilajit. However, it is alsowithin the scope of claims provided herewith for the herbo-mineralformulation to include, as a substitute or additionally, other similarcalcined preparations or minerals without otherwise deterring from theintended function of the herbo-mineral formulation.

In an embodiment, the mineral element includes shilajit in the range of6 to 10 wt %. In another embodiment, the mineral element includes SwarnaMakshika Bhasma is in an amount of 1 to 4 wt %; Mandura Bhasma is in anamount of 2 to 6 wt %; Loha Bhasma is in an amount of 1 to 4 wt %.

Alkali

Further, in an embodiment, the alkali includes at least one alkaliselected from a group consisting of Yavakshara and Sarjakshara.Yavakshara disclosed in the embodiments herein includes an alkali ofHordeum vulgare, and Sarjakshara includes Barilla. In an embodiment, theherbo-mineral formulation disclosed herein includes alkali in an amountin the range of ≤2 wt %.

Salt

In an embodiment, the salt includes at least one salt selected from agroup consisting of Rock salt, Sonchal salt and Black salt. In anembodiment, the herbo-mineral formulation disclosed herein includes thesalt in an amount in the range of 1 to 4 wt %.

The disclosed formulation, in the various embodiments herein, mayfurther include a suitable excipient. The suitable excipients includesolvents, binders, lubricants, herbal carriers, oils and salts that aregenerally known in the art. In a preferred embodiment, the excipientincludes acacia gum.

Further, the amount of herb element and mineral element that may beincluded in the various embodiments of the disclosed formulation may bein the range of 0 to 10 wt %. In an embodiment, the formulation includesCinnamomum camphora (1 to 4 wt %), Acorus calamus (1 to 4 wt %),Saussurea lappa (1 to 4 wt %), Andrographis paniculata (1 to 4 wt %),Tinospora cordifolia (1 to 4 wt %), Cedrus deodara (1 to 4 wt %),Curcuma longa (1 to 4 wt %), Aconitum heterophyllum (1 to 4 wt %),Berberis aristata (1 to 4 wt %), Piper longum (1 to 4 wt %), Plumbagorosea (1 to 4 wt %), Coriandrum sativum (1 to 4 wt %), Emblicaofficinalis (1 to 4 wt %), Terminalia chebula (1 to 4 wt %), Terminaliabellerica (1 to 4 wt %), Piper chaba (1 to 4 wt %), Embelia ribes (1 to4 wt %), Piper chaba (1 to 4 wt %), Piper longum (1 to 4 wt %), Pipernigrum (1 to 4 wt %), Zingiber officinalis (1 to 4 wt %), Commiphoramukul (6 to 10 wt %), Shilajit (6 to 10 wt %), Saccharum officinarum (2to 6 wt %), Swarna Makshika bhasma (1 to 4 wt %), Mandura bhasma (2 to 6wt %), and Loha bhasma (1 to 4 wt %).

In an embodiment, the formulation includes Cinnamomum camphora (1 to 4wt %), Acorus calamus (1 to 4 wt %), Saussurea lappa (1 to 4 wt %),Andrographis paniculata (1 to 4 wt %), Tinospora cordifolia (1 to 4 wt%), Cedrus deodara (1 to 4 wt %), Curcuma longa (1 to 4 wt %), Aconitumheterophyllum (1 to 4 wt %), Berberis aristata (1 to 4 wt %), Piperlongum (1 to 4 wt %), Plumbago rosea (1 to 4 wt %), Coriandrum sativum(1 to 4 wt %), Emblica officinalis (1 to 4 wt %), Terminalia chebula (1to 4 wt %), Terminalia bellerica (1 to 4 wt %), Piper chaba (1 to 4 wt%), Embelia ribes (1 to 4 wt %), Piper chaba (1 to 4 wt %), Piper longum(1 to 4 wt %), Piper nigrum (1 to 4 wt %), Zingiber officinalis (1 to 4wt %), Commiphora mukul (6 to 10 wt %), Shilajit (6 to 10 wt %),Saccharum officinarum (2 to 6 wt %), Yavakshara, (1 to 4 wt %),Sarjakshara (1 to 4 wt %), Swarna Makshika bhasma (1 to 4 wt %), Mandurabhasma (2 to 6 wt %), and Loha bhasma (1 to 4 wt %).

Further, in yet another embodiment, the formulation includes Cinnamomumcamphora (1 to 4 wt %), Acorus calamus (1 to 4 wt %), Saussurea lappa (1to 4 wt %), Andrographis paniculata (1 to 4 wt %), Tinospora cordifolia(1 to 4 wt %), Cedrus deodara (1 to 4 wt %), Curcuma longa (1 to 4 wt%), Aconitum heterophyllum (1 to 4 wt %), Berberis aristata (1 to 4 wt%), Piper longum (1 to 4 wt %), Plumbago rosea (1 to 4 wt %), Coriandrumsativum (1 to 4 wt %), Emblica officinalis (1 to 4 wt %), Terminaliachebula (1 to 4 wt %), Terminalia bellerica (1 to 4 wt %), Piper chaba(1 to 4 wt %), Embelia ribes (1 to 4 wt %), Piper chaba (1 to 4 wt %),Piper longum (1 to 4 wt %), Piper nigrum (1 to 4 wt %), Zingiberofficinalis (1 to 4 wt %), Commiphora mukul (6 to 10 wt %), Shilajit (6to 10 wt %), Saccharum officinarum (2 to 6 wt %), Yavakshara, (1 to 4 wt%), Sarjakshara (1 to 4 wt %), Rock salt (1 to 4 wt %), Sonchal salt (1to 4 wt %), Black salt (1 to 4 wt %), Swarna Makshika bhasma (1 to 4 wt%), Mandura bhasma (2 to 6 wt %), and Loha bhasma (1 to 4 wt %).

In another embodiment, the formulation further includes at least one ofOperculina turpethum, Baliospermum montanum, Cinnamomum tamala,Cinnamomum zeylanica, Elettaria cardamomum, Bamboo manna, Boerhaviadiffusa, Cyperus rotundas, Bauhinia variegate and Holarrhenaantidysenterica in an amount in the range of ≤2 wt %.

Further, the amount of gum acacia may be any amount suitable to performthe activity of an excipient. In an embodiment, the formulation mayinclude gum acacia in the range of 0 to 50 mg per 500 mg of theformulation, preferably 10 wt %.

However, it is apparent that slight variations in the amount of theingredients may be performed without otherwise deterring from theintended function of the herbo-mineral formulation.

The herbo-mineral formulation disclosed herein may be formulated invarious dosage forms such that it is suitable for oral administration.The herbo-mineral formulation may be in the form of tablets, pellets,lozenges, granules, capsules, solutions, emulsions, suspensions, or anyother form suitable for use. In an embodiment, the herbo-mineralformulation is formulated in the form of tablets, preferably 500 mgtablets. For example: Table 1A depicts the quantities of each ingredientin a 500 mg tablet.

Further disclosed herein, is a tablet for treating/preventing/managingRenal disorders. In an embodiment, the tablet is a 500 mg tablet havingherb element, mineral element and an excipient as depicted in Table 1A.

TABLE 1A Each 500 mg tablet includes: SANSKRIT NO. NAME PART USEDSCIENTIFIC NAME QUANTITY 1. Karpura crystals Cinnamomum camphora 10 mg2. Vacha dry root Acorus calamus 10 mg 3. Kushtha dry root Saussurealappa 10 mg 4. Bhunimba dry plant Anrographis paniculata 10 mg 5. Amrtadry stem Tinospora cordifolia 10 mg 6. Devadaru dry heartwood Cedrusdeodara 10 mg 7. Haridra dry rhizome Curcuma longa 10 mg 8. Ativisha dryroot Aconitum heterophylum 10 mg 9. Darvi dry root Berberis aristata 10mg 10. Pippalimula dry root Piper longum 10 mg 11. Chitraka dryroot-pure Plumbago rosea 10 mg 12. Dhanyaka dry fruit Coriandrum sativum10 mg 13. Amalaki dry fruit Emblica officinalis 10 mg 14. Hareetaki dryfruit Terminalia chebula 10 mg 15. Vibhitaki dry fruit Terminaliabellerica 10 mg 16. Chavya dry stem Piper chaba 10 mg 17. Vidanga dryfruit Embelia ribes 10 mg 18. Gajapippali dry root Piper chaba 10 mg 19.Pippali dry fruit Piper longum 10 mg 20. Maricha dry fruit Piper nigrum10 mg 21. Shunthi dry rhizome Zingiber officinalis 10 mg 22. MakshikaIncinerated ore Incinerated Copper pyrite 10 mg 23. Yavakshara alkaliAlkali of Hordeum vulgare 10 mg 24. Sarjakshara alkali Barilla 10 mg 25.Saindhava salt Rock salt 10 mg 26. Souvarchala salt Sonchal salt 10 mgLavana 27 Vida Lavana salt Black salt 10 mg 28. Trivrit dry root dryroot Operculina terpethum  5 mg 29. Danti dry root Baliospermum montanum 5 mg 30. Patraka dry leaves Cinnamomum tamala  5 mg 31. Tvak dry stembark Cinnamomum zeylanica  5 mg 32. Ela dry root Elettaria cardamomum  5mg 33. Vamshalochana Secretion Bamboo manna  5 mg 34. Punarnava Dry rootBoerhavia diffua  5 mg 35. Musta Dry root Cyperus rotundas  5 mg 36.Kanchanara Dry stem bark Bauhinia variegate  5 mg 37. Kutaja Dry stembark Holarrhena antidysenterica  5 mg 38. Sita Extract Saccharumofficinarum-rock 20 mg sugar 39. Mandura Bhasma Incinerated iron Ferrioxidum precipitatum 20 mg rust fuscum 40. Loha Bhasma Incinerated metalIncinerated Iron 10 mg 41. Shilajatu fossil resin Asphaltum 40 mg 42.Shuddha Guggulu oleo-gum-resin Commophora mukul 40 mg 43. Excipient gumGum acacia 50 mg

Embodiments of the disclosed formulation in tablet form were analyzedfor parameters including physicochemical properties such as Tablethardness, Loss on drying, Assay, Disintegration time, Ash value, etc andthe results were noted. Table 2 depicts the results of the analysisperformed to determine the physicochemical properties of an embodimentof the disclosed formulation. In an embodiment, the disclosedformulation tablets have the characteristics as depicted in Table 2. Itwill be apparent to those skilled in the art that many modifications,both to materials and methods, may be practiced without departing fromthe scope of the present invention.

TABLE 2 TEST PARAMETERS SPECIFICATIONS Description Dark brown coloredbiconvex discs Identification Positive for Iron, Calcium Average weight500 mg ± 12.5 mg Uniformity of weight ±2.5% of actual average weightTablet hardness 3.6 kg/cm² Loss on drying  6.2% w/w Methanol solubleextractive 41.2% w/v Chloroform soluble extractive 12.0% w/v Ash value15.2% w/w Average Disintegration time 26 minutes Assay Each tabletcontains, Iron - 4.5 mg, Calcium - 15 mgMethod

Disclosed herein are embodiments of a method of preparing theherbo-mineral formulation. In an embodiment, the method includes,

-   -   levigating bhasmas, Guggulu and shilajit in a grinder;    -   adding herbs, alkali and salts into the grinder; and    -   adding grinding decoction, fresh juice of herbs and gomutra        while continuing grinding to obtain a ground mass.

The bhasmas include at least one of Loha Bhasma, Mandura Bhasma andSwarna makshika bhasma. The mixture of bhasmas, Guggulu and Shilajit maybe in semi-solid form. In an embodiment, the levigation may be performedfor a duration of around 3 hours.

Further, the herbs include finely powdered dried root of Acorus calamus,Saussurea lappa, Aconitum heterophyllum, Berberis aristata, Piperlongum, Piper chaba, Plumbago rosea, Operculina turpethum, Baliospermummontanum Elettaria cardamomum, Boerhavia diffusa and Cyperus rotundas;finely powdered dried fruits of Coriandrum sativum, Emblica officinalis,Terminalia chebula, Terminalia bellerica, Piper longum Embelia ribes,and Piper nigrum; crystals of Cinnamomum camphora; finely powdered driedheartwood of Cedrus deodara; finely powdered dried leaves of Cinnamomumtamala; finely powdered dried whole plant of Andrographis paniculata;finely powdered dried rhizome of Zingiber officinalis and Curcuma longa;finely powdered dried stem of Tinospora cordifolia Saccharum officinarumand Piper chaba; and finely powdered dried stem bark of Cinnamomumzeylanica, Bauhinia variegata and Holarrhena antidysenterica; secretionof Bamboo manna and oleo-gum resin of Commiphora mukul. In anembodiment, finely powdered herbs/herb components may be obtained bypowdering and sieving the herb components through 80 mesh screen.

The grinding decoction is a decoction of herbs that may facilitategrinding. In an embodiment, the grinding decoction includes a decoctionof at least one herb selected from a list consisting of: Cyperusrotundas, Piper longum, Asparagus racemosus, Bauhinia variegate,Oldenlandia umbellata, Hemidesmus indicus, Vetiveria zizanioides,Emblica officinalis, Terminalia chebula, Terminalia bellerica andBoerhavia diffusa. The decoction may be obtained by any method ofdecocting generally known in the field. In an embodiment, the method ofpreparation of grinding decoction further includes, soaking the grindingherbs i.e. powdered dry root of Cyperus rotundas, dried fruit of Piperlongum, dried root of Asparagus racemosus, dried stem bark of Bauhiniavariegata, dried whole plant of Oldenlandia umbellata, dry root ofHemidesmus indicus, dry root of Vetiveria zizanioides, dry fruit ofEmblica officinalis, dry fruit of Terminalia chebula, dry fruit ofTerminalia bellerica and dry root of Boerhavia diffusa; andconcentrating by boiling.

In another embodiment, soaking may be performed by soaking the grindingherbs in 16 parts of water overnight. In a further embodiment,concentrating may be performed by boiling at high temperature,preferably about 80 to 85 degree Celsius, until ⅛th of the liquidremains. Concentration may be confirmed with the help of Brix meter.

Further, once the grinding decoction is added, fresh juice of herbs isadded while continuing grinding. The fresh juice of herbs includes freshjuice of at least one of herb selected from a list consisting of Aloevera, Punica granatum, Cynodon dactylon and Ananas comosus. The freshjuice of herbs may be added obtained by any method generally known inthe field. In an embodiment, fresh juice may be obtained by grinding theherbs either together or separately. Furthermore, once the fresh juiceof herbs is/are added, gomutra (cow urine) is added while grinding iscontinued. In an embodiment, grinding is continued for about 72 hours,preferably at 120 rpm, to obtain a ground mass. In an embodiment, themethod of preparation may further include adding excipient to the groundmass, wherein gum acacia may be added to the ground mass by dissolvingin the grinding decoction while continuing grinding for 3 hours toobtain a semisolid mass. The method of preparation may further includedrying at 50 degree Celsius, preferably in a hot air oven, wetgranulating, punching to obtain 500 mg tablets. FIG. 2 depicts aflowchart for the preparation of fortified tablets. Table 1B depicts theHerb ingredients required for grinding (grinding herbs) in one of thepreferred embodiments.

TABLE 1B List of Grinding herbs Decoction of following herbs: 1. Mustadry root Cyperus rotundas 1 part 2. Pippali dried fruit Piper longum 1part 3. Shatavari dried root Asparagus 1 part racemosus 4. Kanchanaradried stem Bauhinia variegata 1 part bark 5. Parpata dried wholeOldanlandia 1 part plant umbelata 6. Sariva dry root Hemidesmus indicus1 part 7. Usheera dry root Vetiveria 1 part zizanioides 8. Amalaki dryfruit Emblica officinalis 1 part 9. Hareetaki dry fruit Terminaliachebula 1 part 10. Vibhitaki dry fruit Terminalia bellerica 1 part 11.Punarnava dry root Boerhavia diffusa 1 part 12. Jala Water 176 parts 13.Avashesha ⅛ part of water (Reduced to) Fresh juice of following herbs:14. Kumari fresh juice Aloe vera 1 part 15. Dadima fresh juice Punicagranatum 1 part 16. Durva fresh juice Cynodon dactylon 1 part 17.Ananasa frsh fruit juice Ananas comosus 1 part 18. Gomutra Cow urine 1part

The bhasmas that are used in the various embodiments of the disclosedherbo-mineral formulation may be prepared by methods that are generallyknown in the field. Bhasmas may be prepared by selecting genuinestandard minerals as starting material such as Iron rust, Iron, copperpyrite etc; drying in a hot air oven; purifying the mineral bytriturating, quenching, boiling etc; triturating with herbal decoction;preparing into discs; drying of discs; preparing sharavasam puta,subjecting Sharavasam puta to Gaja puta, and powdering of discs oncecooled. In an embodiment, the method is repeated 30 times till bhasma isobtained.

The starting materials used in the preparation of bhasmas may includestandard minerals generally used in the field. In an embodiment, thepreparation of Swarna makshika Bhasma includes Swarna makshika as thestarting material. FIG. 1(a) depicts a flowchart for the preparation ofSwarna Makshika Bhasma using Swarna makshika as the starting material.In another embodiment, the preparation of Mandura Bhasma includes Ironrust as the starting material. FIG. 1(b) depicts a flowchart for thepreparation of Mandura Bhasma using Iron rust as the starting material.In an embodiment, the preparation of Loha Bhasma includes steel iron asthe starting material. FIG. 1(c) depicts a flowchart for the preparationof Loha Bhasma using steel iron as the starting material.

The purification, or shodhana, of the mineral may be performed bygenerally known methods in the field. In an embodiment, the purificationmay be by mixing the starting material with rock salt and lemon juicewherein it is further used in the preparation of Swarna makshika Bhasma.In an embodiment, the purification may be by heating the mineral tillred hot and dipping in 5 different liquid media such as sesame oil,butter milk, cow urine, Kanji (sour medicated rice gruel) and Horse gramdecoction wherein it is further used in the preparation of ManduraBhasma. In another embodiment, the purification may be by quenching themineral in Triphala decoction which is further used in the preparationof Loha Bhasma.

The herbal decoction/juices used for triturating may be any herbaldecoction/juice that is generally used for triturating in thepreparation of bhasmas. For example, the herbal decoction/juice mayinclude triphala, lemon juice, Gomutra (cow's urine) etc. In anembodiment, the herbal decoction includes cow urine and triphaladecoction wherein it is useful in the preparation of Mandura bhasma. Inanother embodiment, the herbal decoction specifically includes TriphalaKashaya (decoction of fruits of Terminalia chebula, Terminalia bellericaand Emblica officinalis) wherein it is useful in the preparation of LohaBhasma. In yet another embodiment, trituration may be performed withlemon juice wherein it is useful in the preparation of Swarna makshikabhasma.

Treatment

Disclosed herein are embodiments of the method oftreating/preventing/managing Renal disorders and associatedcomplications. The embodiments disclosed herein are also instrumental inpreventing the complications associated with kidney diseases such asanemia, electrolyte imbalance, high blood pressure, proteinuria,hyperkalemia, etc.

In an embodiment, the method includes administering to a patient acomposition as described in any of the embodiments disclosed herein. Inan embodiment, the patient may include any individual in need of suchtreatment including ones having/suspected of having Renal disorders orits symptoms. Further, the patient may also include any individualhaving/suspected of having complications associated with Renal disorderssuch as anemia, electrolyte imbalance, high blood pressure, proteinuria,etc.

In a preferred embodiment, the method includes administering to apatient a composition having herb element, mineral element, at least onealkali, at least one salt and suitable excipient, wherein the herbelement includes Cinnamomum camphora (1 to 4 wt %), Acorus calamus (1 to4 wt %), Saussurea lappa (1 to 4 wt %), Andrographis paniculate (1 to 4wt %), Tinospora cordifolia (1 to 4 wt %), Cedrus deodara (1 to 4 wt %),Curcuma longa (1 to 4 wt %), Aconitum heterophyllum (1 to 4 wt %),Berberis aristata (1 to 4 wt %), Piper longum (1 to 4 wt %), Plumbagorosea (1 to 4 wt %), Coriandrum sativum (1 to 4 wt %), Emblicaofficinalis (1 to 4 wt %), Terminalia chebula (1 to 4 wt %), Terminaliabellerica (1 to 4 wt %), Piper chaba (1 to 4 wt %), Embelia ribes (1 to4 wt %), Piper chaba (1 to 4 wt %), Piper longum (1 to 4 wt %), Pipernigrum (1 to 4 wt %), Zingiber officinalis (1 to 4 wt %), Commiphoramukul (6 to 10 wt %), Saccharum officinarum (2 to 6 wt), and/or at leastone of herb selected from Operculina turpethum, Baliospermum montanum,Cinnamomum tamala, Cinnamomum zeylanica, Elettaria cardamomum, Bamboomanna, Boerhavia diffusa, Cyperus rotundas, Bauhinia variegata andHolarrhena antidysenterica, (≤2 wt %); the mineral element includesShilajit (6 to 10 wt %) and at least one of bhasma selected from Mandurabhasma (2 to 6 wt %) and Loha bhasma (1 to 4 wt %); at least one alkaliselected from a group consisting of Yavakshara (≤2 wt %) and Sarjakshara(≤2 wt %); and at least one salt selected from a group consisting ofRock salt (1 to 4 wt %), Sonchal salt (1 to 4 wt %) and Black salt (1 to4 wt %).

In an embodiment, the disclosed formulation may be used such that itacts as at least one of anti-lipid, free radical scavenging,hypolipidemic and nephro-protective agent.

The disclosed method of treatment may be used as a primary line oftreatment or as an adjunct to other treatment methods for kidneydisorders.

The patient may be administered a therapeutically effective amount ofthe embodiments of the disclosed formulation. The therapeuticallyeffective amount may vary depending on the patient. In an embodiment,the therapeutically effective amount is 500 to 1000 mg administered oneto three times a day.

Embodiments of the disclosed formulation were subjected to acute oraltoxicity study, and a study to check its effect on behavior and nervoussystem. The studies showed that the embodiments of the formulation arefree from toxicity even at a dose of 6000 mg/kg weight which was themaximum possible dose. It was also found to have no harmful effects onbehavioral and nervous system.

Embodiments of the Disclosed formulation (also referred as Test productor Test drug) were further evaluated for efficacy of nephron-protectiveactivity by preclinical and clinical studies, as described hereunder byway of examples. Embodiments are further described by reference to thefollowing examples by way of illustration only and should not beconstrued to limit the scope of the claims provided herewith. It will beapparent to those skilled in the art that many modifications, both tomaterials and methods, may be practiced without departing from the scopeof the claims.

Example 1: Preclinical Study

The aim of this study was to analyze the effect of Test drug againstGentamicin induced Nephrotoxcity in male Sprague dawley rats.

Experiment Details:

24 Sprague dawley rats between the range of 200-300 g were selected andacclimatized for a period of 5 days to the laboratory condition. Afteracclimatization, animals were randomized into four groups (Group I-IV)consisting of six animals per group. Group I and II animals receivedvehicle (0.5% CMC); group III and IV received Test drug at 100 and 200mg/kg b.wt., p.o., respectively for a period of 14 days. After 1 hr ofdrug administration, all experimental animals received Gentamicin (100mg/kg; i.p) intraperitoneally once daily except those from normalcontrol group for 14 days. Weekly drug dosage was adjusted based on therespective week body weight of animals.

Clinical signs of toxicity, morbidity and mortality were observed dailytill the day of necropsy. Body weight was recorded once before dosingand on day 7 and 14. Urine output and Urine biochemistry was analyzed onday 7 and 14 by individually housing the animals in metabolic cages.Biochemistry and electrolytes in serum were also analyzed at the end oftreatment and necropsy. A renal weight change and gross pathology ofliver and renal tissues were observed.

Treatment related death and clinical signs of toxicity were not foundbetween the experimental animals. Significant decrease in body weightwas observed on day 14 in Group II. Group III and Group IV animalsprevented loss of body weight in comparison with Group II.

No change in color, pH, and Specific gravity, excretion of blood,nitrites, bilirubin, and ketones in urine were noted between Group I,Group II, III and IV animals. Traces of glucose, micro albumin, protein,albumin, urobilinogen and leucocytes were observed in urine of group II,III and IV at day 14.

Group II animals also showed significant elevation in urea, creatinine,uric acid, alkaline phosphatase, gamma glutamyl transferase wereobserved at day 14 in urine and blood when compared to Group I. GroupIII and IV (Test drug, 100 and 200 mg/kg) significantly attenuates theselevels a dose dependent manner.

No significant change in serum electrolytes such as potassium, pH,sodium were observed between the treated groups when compared to GroupI. Significant elevation in Calcium was observed in Group II which wassignificantly attenuated by Group IV to normal range.

Lipid peroxidation was significantly (p<0.05) augmented by gentamicinadministration with concomitant decrease in antioxidants, Super oxidedismutase, reduced Glutathione and Glutathione peroxidase in Group II.Group III and IV animals reverse these alterations in a dose dependentmanner.

Gentamycin has not shown any gross pathological changes between thegroups. However, histopathology of renal tissues reveal mild to moderatedegree of lesions such as tubular degeneration, tubular cell necrosisetc, in Group II animals which confirms the induction of nephrotoxicityin rats. Remarkable decrease in severity and incidence of lesions inrenal tissues was observed in Group IV animals (Test drug treated at 200mg/kg b.wt) when compared to animals of Gentamycin treated group andconcurrent control.

Histology of Livers of animals treated with Test drug treated at 100 and200 mg/kg b.wt reveals decrease in severity of inflammation in group IIanimals.

Altogether, the present study reports the potential nephrotoxicityproduced by Gentamicin in rats through the excretion of glucose,protein, micro albumin, leucocytes and urobilinogen in urine. Test drugtreatment has exasperated to counteract these responses at both doselevels with antioxidants defense. The result of this study demonstratedthat Test drug has potent nephroprotective action uponGentamycin-induced renal damage in rats and possessed antilipidperoxidative and free radical scavenging activities.

Experiment Details:

Materials:

Species/Strain: Rat/Sprague Dawley

Sex: Male

Body weight at the time: 200-260 g of Dosing

Acclimatization: Minimum five days under laboratory conditions.

Chemicals used:

Gentamicin Tris Hcl Buffer (pH 7.2) Anaesthetic ether Sodiumpyrophosphate buffer (0.025M) Tri Chloro acetic acid NADH (780 μM)Phosphate buffer (pH:8) Saline Dithio dinitro benzoic acid (DTNB)Standard malondialdehyde in 0.2M Phenazonium Metho Sulphate (PMS) Sodiumazide (186 μM) Nitro Blue Tetrazolium chloride Hydrogen Peroxide (NBT)(300 μM) Reduced glutathione Glacial acetic acid Thiobarbituric acid(0.8%, TBA) in n-butanol 0.5N HCl. Butylated Hydroxyl Toluene (BHT)0.05% Potassium•EDTA in methanol.

Consumables

Surgicals Rat Cages with grid Micro Tips-10 μl & 1000 μl Husk Eppendrofvial 2 ml Syringes Capillaries (heparinised) Oral gavage Polypropylenewater bottle

Instrument/Equipment Used:

Below mentioned equipment's were periodically calibrated as applicable;

Anaesthetic chamber Semi automated analyser Animal weighing BalanceFully automated analyser Analytical weighing balance Microplate readerMetabolic cage Micropipette - 100-1000 μl, 20-200 μl Tissue homogenizerRefrigerated centrifuge Cyclomixer Electrolyte analyser

Experimental Conditions:

-   Environmental conditions: Temperature, humidity and air exchange    were maintained in the range of 19-23° C., 30-70% and 12-15 air    changes per hour respectively. The animals were provided with    photoperiod of 12 hours artificial light and 12 hours dark.-   Housing: Animals were housed in groups polypropylene cages with    deducted and bedding material-   Diet & Water: Animals were provided with commercially available    laboratory rodent feed and reverse osmosis water.    Procedure:

Grouping and Dosing: Animals were grouped six per cage in the followingmanner. Table 3 depicts the grouping and dosing of animal.

TABLE 3 Total Group Treatment animals I Normal Control 0.5% CMC 6 IIPositive Gentamicin (100 mg/kg/day in i.p) + 6 Control 0.5% CMC III Testdrug Test drug (100 mg/kg, p.o) + Gentamicin 6 (100 mg/kg/day in i.p) IVTest drug Test drug (200 mg/kg, p.o) + Gentamicin 6 (100 mg/kg/day ini.p

Induction: All experimental animals received Gentamicin (100 mg/kg; i.p)except normal control 1 hr after the drug treatment for 14 days.

Dose Formulation: The test and reference drug was freshly prepared withappropriate volume of 0.5% CMC prior to administration.

Dosage:

-   -   10 ml/kg were the dose volume    -   Dosage of administration of test substance was adjusted based on        the weekly body weight.    -   Test drug, reference drug, vehicle formulations were        administered orally to respective animals via intragastric tube        for a period of 14 days.

Urine Collection:

-   -   On day 7 and 14, animal will be housed for 24 hr in metabolic        cages for urine collection.    -   Urine output was measured and used for analysis using urine        strip.

Blood Collection:

-   -   On day 15, animals were anesthetized using Isofluorene for blood        collection. Blood was collected in an Eppendorf vials and kept        in slanting position at room temperature for 15-30 minutes for        serum separation.    -   Vials were centrifuged at 3500 rpm for 10 minutes for separation        and used for Biochemical analysis.

Histopathology:

-   -   Organ kidney and liver were collected for histopathological        process

Data Analysis: All values were expressed individually as Mean±S.E.M.Statistical analyses were performed on the different conditions usingone-way ANOVA (F value), followed by the post-hoc analysis (Tukeymultiple comparison test). p<0.05 and p<0.01 are considered asstatistically significant.

Observation:

Mortality & Morbidity: Mortality and morbidity of the animals waschecked daily.

Clinical Signs: Clinical signs were observed in experimental animalsthroughout the study.

Body Weight: Body weight was recorded on day 0, 7 and 14.

Urine parameters: pH, specific gravity, Bilirubin, blood, nitrites,glucose, microalbuminuria (MALB), Albumin, protein, ketones andleucocytes in urine were quantified using urine analyser via urinary dipstrips. Urea, Creatinine, Uric acid and Albumin content and the activityof enzymes such as alkaline phosphatase (ALP), gamma glutamyltransferase (GGT) and acid phosphatase (ACP) in urine was analyzed usingBio-Systems Diagnostics Pvt. Ltd with Semi Automated biochemicalanalyser.

Blood parameters: Serum electrolytes (Calcium, Potassium, pH, sodium)were analyzed using Cornley Acculyte 5P electrolyte analyzer. Blood ureanitrogen (BUN), Creatinine, uric acid, total protein, albumin, globulin,alkaline phosphatase (ALP), gamma glutamyl transferase (GGT) and acidphosphatase (ACP) activities were analyzed using Bio-Systems DiagnosticsPvt. Ltd as per kit manual.

Oxidative stress markers: About 500 mg of renal tissues were homogenizedin 5 mL KCl [10 mM] phosphate buffer (1.15%) with ethylene-diamine tetraacetic acid (EDTA, pH 7.4) and centrifuged at 5, 000 rpm for 60 min. Thesupernatant were used to measure oxidative stress markers such assuperoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH),lipid peroxides (TBARS), and total protein (TP) using Multi platereader.

Tissue Lipid Peroxide (LPO, TBARS) Level: Tissue lipid peroxide (LPO)level was determined as TBA-reactive substances according to the methodof Ohkawa et al (1979). To 1.0 ml of homogenate, 3.0 ml of 0.8%, TBA HClreagent and 0.5 ml of Butylated Hydroxyl Toluene (BHT) 0.05% in methanolwere added and mixed thoroughly. The mixture was kept in a boiling waterbath for 15 minutes. After cooling, the tubes were centrifuged at 1000 gfor 10 minutes and the supernatant was taken for the measurement. Astandard solution of malondialdehyde at various concentrations wastreated in a similar manner. The absorbance of pink chromophore was readat 535 nm against a reagent blank. Values were expressed as microgram/gmtissue.

Total protein: Protein content in tissue was measured by Lowry et al.,1951. To 0.025 ml of homogenate, 0.5 ml of Lowry reagent was added andmixed thoroughly. The mixture was kept t room temperature for 15minutes. A standard solution of BSA at various concentrations wastreated in a similar manner. The absorbance of blue chromophore was readat 540 nm against a reagent blank. Values were used to express theenzyme activity.

Enzymic and Non-Enzymic Antioxidants

Superoxide Dismutase: Superoxide dismutase activity was assayed by themethod of Kakkar et al., 1984. Superoxide dismutase was assayed bytaking 0.05 ml of homogenate followed by addition of 0.3 ml of sodiumpyrophosphate buffer, 0.025 ml of Phenazonium Metho Sulphate (PMS; 186μM) and 0.075 ml of Nitro Blue Tetrazolium chloride (NBT, 300 Thereaction was started by addition of 0.075 ml of NADH (780 Afterincubation at 300 C for 90 seconds, the reaction was stopped by additionof 0.25 ml glacial acetic acid. Then the reaction mixture was stirredvigorously and shaken with 2.0 ml of n-butanol. The mixture was allowedto stand for 10 minutes and centrifuged. 1.5 ml of n-butanol alone wasserved as blank. The colour intensity of the chromogen was read at 560nm.

Calculation: Enzyme Activity (1 Unit)=50% Inhibition/Minute.

Glutathione Peroxidase: Glutathione peroxidase activity was estimated bythe method of Rotruck et al., (1973). Activity of Glutathione peroxidase(GPx) was assayed by taking 200 μl of 0.4 mM Tris HCl Buffer (pH 7.2),200 μl K.EDTA (0.4 mM) along with 100 μl of sodium azide (10 mM) and 200μl of enzyme preparation and mixed well. Thereafter, 200 μl of reducedglutathione solution (2 mM) followed by 0.1 ml H2O2 were added. Theoverall reaction was arrested by adding 0.5 ml of 10% TCA. Thenon-enzymatic reaction rate was correspondingly assessed by replacingthe enzyme sample by buffer. The precipitate was removed bycentrifugation at 4000 rpm for 10 minutes. The remaining reducedglutathione in the supernatant was determined by adding 1.0 ml of 0.6 mMDithio dinitro benzoic acid (DTNB) in 0.2 M Phosphate buffer (pH 8.0).The absorbance was read at 412 nm using an UV/Visible spectrophotometer.The content of GSH was calculated using standard graph of GSH. Theresults are expressed in microgram of GSH consumed per minute per mgprotein.

Catalase: Catalase activity was estimated by the method of Sinha AK1972. Two set of tubes were arranged and labelled as Test and Control.To ‘Test’, 0.1 ml of homogenate and 0.5 ml of Phosphate buffer was addedand the reaction was initiated by adding 0.4 ml of H2O2. To Control, 0.6ml of Phosphate buffer and 0.4 ml of H2O2 were added. The reactionmixture was incubated at 5 minutes at room temperature. Afterincubation, 2 ml of Dichromatic acetic acid was added and boiled for 10minutes. 0.1 ml of homogenate was added to control tubes. The chromicacetate thus produced is measured colorimetrically at 570 nm. Theactivity of CAT was expressed as units/mg protein. One unit of CATactivity represents the amount of enzyme that destroys 1 μmole H2O2/min.

Reduced Glutathione (GSH): Reduced glutathione content was measured bythe method of Moren et al., 1979. 0.25 ml of homogenate was added toequal volume of ice cold 5% TCA to precipitate the protein present inthe tissue. The precipitate was removed by centrifugation at 4000 rpmfor 10 minutes. To 1 ml aliquot of supernatant, 0.25 ml of phosphatebuffer, pH 8.0 and 0.5 ml of 0.6 mM Dithio dinitro benzoic acid (DTNB)in 0.2 M Phosphate buffer (pH 8.0). was added and mixed well. Theabsorbance was read at 412 nm using an UV/Visible spectrophotometer. Thecontent of GSH was calculated using standard graph of GSH. The resultsare expressed in micromoles of GSH per gram tissue.

Necropsy: At the end of the study, all surviving animals were euthanizedby CO2 asphyxiation and all animals were subjected to detailed grossnecropsy which includes external surface of the body and grossexamination of external orifices, the cranial, thoracic and abdominalcavities and their contents.

Histopathology: From the collected organs, adequate amount of kidney andliver tissues were placed in formalin filled container (1:10 ratio) forfixation, approx 3-5 mm thick tissues were trimmed, processed inalcohol, xylene and impregnate in paraffin. Processed tissues wereembedded in paraffin block, sectioned at 3-5 micron and stained with H &E for Histopathology examination.

The following grading system was used for histopathology evaluation inthe study:

1—Minimal

2—Mild

3—Moderate

4—Marked

5—Severe

Results and Discussion:

Mortality &Morbidity: No morbidity or mortality was observed inexperimental animals throughout the study.

Clinical Signs: No abnormal clinical signs were observed in experimentalanimals throughout the study. All the animals were found normal.

Body Weight: Significant body weight change was not observed on day 7between the control and treatment groups. However, significant (p<0.05)decrease in body weight was observed in Group II on day 14 when comparedto Group I. Though slight increase in body weight was observed in Testdrug treated rats (Group III and IV), were insignificant and notcomparable to normal control (Group I). Table 4 depicts the effect ofTest drug on Body Weight Changes in Nephrotoxicity Induced Rats

Urine parameters: Urinalysis (UA) is used as a screening tool to detectsubstances or cellular material in the urine associated with renaldysfunction or urinary tract infections (UTI). Significant increase inurine output was noted at day 7 and 14 of group II animals when comparedto group I. But there was no significant change in urine output wasnoted at day 7 and day 14 of group IV when compared to group II. Table 5depicts the effect of Test drug Treatment on Urine Output InNephrotoxicity Induced Rats.

No change in color, pH and Specific gravity between control and treatedanimals. There were no excretion of blood, nitrites, bilirubin, ketoneswere noted in control and treated animals.

Trace amount of glucose (5.6 mmol/L), micro albumin, protein,significant (p<0.01) level of albumin, urobilinogen and leucocytes wereobserved in urine of group II, III and IV at day 14 and no suchelimination was noted in Group I. The result shows that gentamicininduction causes slight damage in renal tissues which in turn reflectsthe excretion of Glucose, protein, micro albumin, leucocytes andurobilinogen. This may be due to the increased renal tubular secretion,increased glomerular filtration, obstruction in the urinary tract andreduced glucose reabsorption in renal tubular disease. Test drugtreatment has counteracted these responses which was insignificant.Table 6 depicts the effect of Test drug Treatment on Urinary ParametersIn Nephrotoxicity Induced Rats.

On the other hand, significant elevation in urea, creatinine, uricacid,alkaline phosphatase (ALP), gamma glutamyl transferase (GGT) wereobserved at day 14 in Group II animals when compared to Group I. Testdrug at two dose levels significantly attenuates these levels whencompared Group II. Table 7 depicts the effect of Test drug Treatment onUrine Biochemistry in Nephrotoxicity Induced Rats. Table 8 depicts theeffect of Test drug Treatment on Organ Weight in Nephrotoxicity InducedRats

Blood parameters: Significant elevation in blood urea nitrogen (BUN),Creatinine, uric acid, alkaline phosphatase (ALP), gamma glutamyltransferase (GGT) were observed in Group II animals when compared toGroup I. Group III and IV (Test drug at two dose levels) attenuates GGTand uric acid levels significantly when compared with group II and alsoreduced the urea and creatinine, the levels were insignificant. Table 9depicts the effect of Test drug Treatment on Blood Biochemistry InNephrotoxicity Induced Rats.

No significant change in serum electrolytes such as potassium, pH,sodium were observed between the treated groups. Significant elevationin Calcium was observed in Group II which was significantly attenuatedto normal by Test drug high dose treatment. As reported earlier, calciumhad a relation with Gentamycin. It impairs calcium transport in therenal tubules (Arphitha et al 2008) and increases the calcium level inserum. Table 10 depicts the Effect of Test drug Treatment on SerumElectrolytes in Nephrotoxicity Induced Rats

Oxidative stress markers: Lipid peroxidation in the renal tissues isassessed by the accumulation of MDA, was significantly (p<0.05)augmented by gentamicin administration with concomitant decrease inantioxidants, Super oxide dismutase, reduced Glutathione and Glutathioneperoxidase Test drug treatment significantly maintained the lipidperoxidation (p<0.05) and glutathione (p<0.01) in gentamycin inducedrats nearer to normal. Similarly, Test drug treatment dose dependentmanner amplified the super oxide dismutase and glutathione peroxidaselevels which were not significant. Table 11 depicts the effect of Testdrug Treatment on Renal Stress Marker In Nephrotoxicity Induced Rats

Gross Pathology: During necropsy, gross lesions like discoloration,pale, pitted surface, bilateral, was observed in kidneys of Group IIanimals. Any gross lesions were not noticed in other organs in any ofthe study animals.

Histopathology: Kidneys of control animals treated with vehicle alonerevealed tubular degeneration and or regeneration, tubular dilatation,and inflammatory cells infiltration but only at within normal limit tominimal severity. Kidneys of Gentamycin (100 mg/kg body weight) inducedanimals revealed classical nephrotoxicity induced histopathologicallesions viz., tubular degeneration and/or regeneration, tubulardilatation, tubular cell vacuolation, tubular cell necrosis andinflammatory cells infiltration with higher severity ranging from mildto moderate degree. Table 12 depicts the Individual AnimalHistopathological Findings.

Animals from both groups treated with Test drug did show protectiveeffect against gentamycin induced toxicity. However, remarkable effecti.e. decreases in severity and incidence of lesions in the kidneys ofanimals was observed in Test drug treated at 200 mg/kg b.wt. compared toanimals treated with Test drug (100 mg/kg b.wt.). Table 13 depicts theSummary of Grading System for Histopathology Evaluation of Kidney

Histopathology of liver though not remarkable but revealed inflammatorycells infiltration with minimal severity in 3/6 animals from gentamycintreatment group which also appeared to be decrease to 1/6 animalstreated with Test drug at 200 mg/kg b.wt. Table 14 depicts the Summaryof Grading System for Histopathology Evaluation of Liver.

FIG. 3 represents the histopathological observations of Kidneys of GroupI and Group II. FIG. 4 represents the histopathological observations ofKidneys of Group III and Group IV. FIG. 5 represents thehistopathological observation of Liver OF Group I, Group II, Group IIIand Group IV.

All other histopathological findings were either spontaneous and orbackground changes, related to agonal changes, routinely observed inrats of this age.

Tabulation of Results is as Follows:

TABLE 4 Effect of Test drug on Body Weight Changes in NephrotoxicityInduced Rats Body weight (g) Treatment Animal No Day 0 Day 7 Day 14Normal control 1 257 284 242 0.5% CMC 2 238 257 234 3 269 300 247 4 238269 252 5 242 216 210 6 244 271 251 Mean ± SEM 243.00 ± 7.92 266.16 ±11.68 239.33 ± 6.45 Gentamicin 7 230 184 156 (100 mg/kg) 8 213 215 178i.p 9 219 295 231 10 222 231 187 11 218 297 245 12 229 233 191 Mean ±SEM 221.80 ± 2.70 242.50 ± 18.37   198.00 ± 13.70^(#) Test drug 13 283291 253 (100 mg/Kg) 14 233 243 212 15 206 188 165 16 209 203 225 17 232235 198 18 250 258 182 Mean ± SEM  235.50 ± 11.60 236.33 ± 15.22  205.83± 12.81 Test drug 19 252 252 252 (200 mg/Kg) 20 226 235 205 21 223 242199 22 232 242 204 23 237 267 209 24 216 219 189 Mean ± SEM 231.00 ±5.14 242.83 ± 6.58  209.67 ± 8.92 The results are expressed inIndividual as well as Mean ± SEM (n = 6); Statistical analysis was doneusing graph pad prism 5.0 version and Tukey post hoc test was performed.^(#)p < 0.05, ##p < 0.01 compared with Normal control: p < 0.05 (*) &0.01 (**) compared with Gentamicin (100 mg/kg) i.p

TABLE 5 Effect of Test drug Treatment on Urine Output In NephrotoxicityInduced Rats. Urine Out Put Urobilinogen (ml) (umol/L) Treatment AnimalNo 7 14 7 14 0.5% CMC 1 4.00 6.00 34 3.4 2 4.00 6.00 34 3.4 3 8.80 6.4034 3.4 4 7.20 5.60 34 3.4 5 12.00 8.00 34 3.4 6 12.00 8.00 34 3.4 Mean ±SEM 8.00 ± 1.48 6.67 ± 0.43 34.00 3.40 Gentamicin (100 mg/kg) i.p 711.20 4.00 34 34 8 8.80 4.00 34 34 9 26.00 9.20 34 34 10 22.00 8.80 3434 11 12.80 10.00 34 34 12 14.00 10.00 34 34 Mean ± SEM  15.80 ±2.74^(##) 7.67 ± 1.17 34.00 34.00 Test drug (100 mg/Kg) 13 28.00 22.4034 34 14 28.00 21.60 34 34 15 20.40 14.00 34 34 16 19.60 14.00 34 34 1725.20 10.00 34 34 18 26.00 12.00 34 34 Mean ± SEM  24.53 ± 1.51**  15.67± 2.09** 34.00 34.00 Test drug (200 mg/Kg) 19 12.80 14.80 34 34 20 29.6015.20 34 34 21 14.00 7.20 34 34 22 14.00 6.80 34 34 23 16.40 4.00 34 3424 16.80 4.00 34 34 Mean ± SEM 17.27 ± 2.55  8.67 ± 2.08 34.00 34.00The results are expressed in Individual values as well as Mean±SEM(n=6); Statistical analysis was done using graph pad prism 5.0 versionand Tukey post hoc test was performed.

TABLE 6 Effect of Test drug Treatment on Urinary Parameters InNephrotoxicity Induced Rats BIL BLD GLUCOSE MALB Animal (umol/L)(Ery/μL) NIT (mmol/L) (g/L) Treatment No 7 14 7 14 7 14 7 14 7 14 0.5%CMC 1 Negative Negative Negative Negative Negative Negative NegativeNegative >0.15 Negative 2 Negative Negative Negative Negative NegativeNegative Negative Negative >0.15 Negative 3 Negative Negative NegativeNegative Negative Negative Negative Negative >0.15 Negative 4 NegativeNegative Negative Negative Negative Negative Negative Negative >0.15Negative 5 Negative Negative Negative Negative Negative NegativeNegative Negative >0.15 Negative 6 Negative Negative Negative NegativeNegative Negative Negative Negative >0.15 Negative Gentamicin 7 NegativeNegative Negative Negative Negative Negative Negative 5.6 >0.15 >0.15(100 mg/kg) 8 Negative Negative Negative Negative Negative NegativeNegative 5.6 >0.15 >0.15 i.p 9 Negative Negative Negative NegativeNegative Negative Negative 5.6 >0.15 >0.15 10 Negative Negative NegativeNegative Negative Negative Negative 5.6 >0.15 >0.15 11 Negative NegativeNegative Negative Negative Negative Negative 5.6 >0.15 >0.15 12 NegativeNegative Negative Negative Negative Negative Negative 5.6 >0.15 >0.15Test drug 13 Negative Negative Negative Negative Negative Negative 5.65.6 >0.15 >0.15 (100 mg/Kg) 14 Negative Negative Negative NegativeNegative Negative 5.6 5.6 >0.15 >0.15 15 Negative Negative NegativeNegative Negative Negative Negative 5.6 >0.15 >0.15 16 Negative NegativeNegative Negative Negative Negative Negative 5.6 >0.15 >0.15 17 NegativeNegative Negative Negative Negative Negative Negative 5.6 >0.15 >0.15 18Negative Negative Negative Negative Negative Negative Negative5.6 >0.15 >0.15 Test drug 19 Negative Negative Negative NegativeNegative Negative 5.6 5.6 >0.15 >0.15 (200 mg/Kg) 20 Negative NegativeNegative Negative Negative Negative 5.6 5.6 >0.15 >0.15 21 NegativeNegative Negative Negative Negative Negative Negative 5.6 >0.15 >0.15 22Negative Negative Negative Negative Negative Negative Negative5.6 >0.15 >0.15 23 Negative Negative Negative Negative Negative NegativeNegative 5.6 >0.15 >0.15 24 Negative Negative Negative Negative NegativeNegative Negative 5.6 >0.15 >0.15 KET PRO LEU Animal SG pH (umol/L)(g/L) (Lue/μL) Treatment No 7 14 7 14 7 14 7 14 7 14 0.5% CMC 1 1.0251.030 5.1 6 Negative Negative 0.3 Negative Ca15 Negative 2 1.025 1.0305.1 6 Negative Negative 0.3 Negative Ca15 Negative 3 1.025 1.030 7.5 60.5 Negative 3 Negative Ca15 Negative 4 1.025 1.030 7.5 6 0.5 Negative 3Negative Ca15 Negative 5 1.015 1.030 6 6 0.5 Negative 0.3 Negative Ca15Negative 6 1.015 1.030 6 6 0.5 Negative 0.3 Negative Ca15 Negative Mean± SEM 1.02 ± 1.03 ± 6.20 ± 6.00 ± 0.50 ± Negative 1.20 ± 0.00 ± Ca15Negative 0.00 0.00 0.44 0.00 0.00 0.57 0.00 Gentamicin 7 1.025 1.030 5.55.5 0.5 Negative 1 1 Ca15 Ca15 (100 mg/kg) 8 1.025 1.030 5.5 5.5 0.5Negative 1 1 Ca15 Ca15 i.p 9 1.015 1.030 6 5.5 0.5 Negative 0.3 1 Ca15Ca15 10 1.015 1.030 6 5.5 0.5 Negative 0.3 1 Ca15 Ca15 11 1.02  1.0306.5 5.5 0.5 Negative 1 1 Ca15 Ca15 12 1.02  1.030 6.5 5.5 0.5 Negative 1I Ca15 Ca15 Mean ± SEM 1.02 ± 1.03 ± 6.00 ± 5.50 ± 0.50 ± Negative 0.77± 1.00 ± Ca15 Ca15 0.00 0.00 0.18 0.00 0.00 0.15 0.00^(#) Test drug 131.025 1.015 6 6 0.5 0.5 3 1  Ca 70 Ca70 (100 mg/Kg) 14 1.025 1.015 6 60.5 0.5 3 1  Ca 70 Ca70 15 1.015 1.015 6 6 0.5 0.5 0.3 1 Ca15 Ca70 161.015 1.015 6 6 0.5 0.5 0.3 1 Ca15 Ca70 17 1.015 1.015 5 6 0.5 0.5 0.3 1Ca15 Ca70 18 1.015 1.015 5 6 0.5 0.5 0.3 1 Ca15 Ca70 Mean ± SEM 1.02 ±1.02 ± 5.67 ± 6.00 ± 0.50 ± 0.50 ± 1.20 ± 1.00 ± Ca30 ± Ca70 0.00 0.000.21 0.00 0.00 0.00 0.57 0.00 11.60 Test drug 19 1.03  1.025 6.5 5.5 0.5Negative 1 3 Ca15 Ca70 (200 mg/Kg) 20 1.03  1.025 6.5 5.5 0.5 Negative 13 Ca15 Ca70 21 1.03  1.025 6 5.5 0.5 Negative 1 3 Ca15 Ca70 22 1.03 1.025 6 5.5 0.5 Negative 1 3 Ca15 Ca70 23 1.015 1.025 6.5 5.5 0.5Negative 1 3 Ca15 Ca70 24 1.015 1.025 6.5 5.5 0.5 Negative 1 3 Ca15 Ca70Mean ± SEM 1.03 ± 1.03 ± 6.33 ± 5.50 ± 0.50 ± Negative 1.00 ± 3.00 ±Ca15 Ca70 0.00 0.00 0.11 0.00 0.00 0.00 0.00**

The results are expressed in Individual values as well as Mean±SEM(n=6); Statistical analysis was done using graph pad prism 5.0 versionand Tukey post hoc test was performed. # p<0.05, ##p<0.01 compared withNormal control: p<0.05 (*) & 0.01 (**) compared with Gentamicin (100mg/kg) i.p

TABLE 7 Effect of Test drug Treatment on Urine Biochemistry inNephrotoxicity Urea Creatinine ACP Albumin Animal (mg/ml) (mg/ml) (U/L)(mg/dl) Treatment No 7 14 7 14 7 14 7 14 0.5% CMC 1 21.61 20.57 0.350.98 50.00 60.00 1.23 1.31 2 15.57 22.66 0.4 1.195 49.00 49.00 1.13 1.153 14.25 42.62 1.55 0.7 58.00 58.00 1.12 1.14 4 28.65 24.56 0.66 0.8951.00 41.00 1.64 1.74 5 25.80 29.9 1.5 1.24 38.00 48.00 1.11 1.41 672.50 30.65 0.605 0.78 43.00 53.00 1.28 1.48 Mean ± SEM 29.73 ± 28.49 ±0.84 ± 0.96 ± 48.17 ± 51.50 ± 1.25 ± 1.37 ± 8.85 3.26 0.22 0.09 2.822.86 0.08 0.09 Gentamicin 7 46.375 66.7 1.62 1.13 61.00 56.00 1.91 2.91(100 mg/kg) 8 44.565 60.965 1.17 1.56 53.00 75.00 0.83 2.93 i.p 9 32.3549.345 1.055 1.945 55.00 73.00 1.54 1.44 10 67.605 51.21 1.39 2.28 51.0045.00 1.18 1.18 11 48.48 59.8 0.945 1.67 54.00 66.33 1.89 1.99 12 49.552.26 1.59 1.28 61.00 72.33 2.02 2.02 Mean ± SEM 48.15 ± 56.71 ± 1.30 ±1.64 ± 54.17 ± 64.61 ± 1.56 ± 2.08 ± 4.65^(##) 2.78^(##) 0.12 0.17* 1.514.83 0.19 0.38^(##) Test drug 13 36.255 40.685 0.785 0.67 46.00 51.000.88 0.89 (100 mg/Kg) 14 39.77 32.68 0.615 1.03 65.00 43.00 1.66 0.92 1533.13 27.105 0.855 0.635 35.00 46.00 0.67 0.55 16 30.825 26.45 0.7150.995 51.00 50.00 1.66 1.03 17 65.8 33.86 1.535 1.23 56.33 64.00 0.771.11 18 55.105 30.06 1.38 0.815 62.33 71.00 1.66 1.99 Mean ± SEM 43.48 ±31.81 ± 0.98 ± 0.90 ± 52.61 ± 54.17 ± 1.22 ± 1.08 ± 5.68 2.14* 0.160.09* 4.54 4.47 0.20 0.20** Test drug 19 26.35 31.825 1.37 0.505 44.3354.33 0.90 0.91 (100 mg/Kg) 20 24.01 37.57 1.22 0.645 56.33 56.33 1.680.94 21 26.225 27.425 1.7 0.6 45.00 63.00 0.73 0.66 22 35.105 33.615 1.60.88 41.00 45.00 1.68 1.05 23 49.005 32.15 1.66 0.875 56.33 66.33 0.791.13 24 46.555 56.215 1.445 1.055 52.00 62.00 1.68 2.01 Mean ± SEM 34.54± 36.47 ± 1.50 ± 0.76 ± 49.17 ± 57.83 ± 1.24 ± 1.12 ± 4.48 4.17* 0.080.09* 2.70 3.14 0.20 0.19** ALP g-GT Uric acid Animal (U/L) (U/L)(mg/dl) Treatment No 7 14 7 14 7 14 0.5% CMC 1 1.00 1.00 37.00 27.006.69 6.09 2 0.00 0.00 36.00 56.00 9.25 9.15 3 0.00 0.00 34.00 64.00 8.788.38 4 1.00 1.00 40.00 50.00 6.00 6.00 5 1.00 1.00 37.00 37.00 6.0010.44 6 1.00 1.00 39.00 39.00 9.00 9.00 Mean ± SEM 0.67 ± 0.67 ± 37.17 ±45.57 ± 7.62 ± 8.18 ± 0.21 0.21 0.87 5.17 0.63 0.73 Gentamicin 7 1.001.00 13.00 13.00 12 13.37 (100 mg/kg) 8 1.00 1.00 26.00 26.00 13 14.86i.p 9 1.00 2.00 23.00 23.00 12.12 13.11 10 1.00 3.00 26.00 36.00 12.1312.30 11 1.00 1.00 34.00 34.00 11.13 11.32 12 1.00 1.00 19.00 29.0011.99 10.99 Mean ± SEM 1.00 ± 1.50 ± 23.50 ± 26.83 ± 12.06 ± 12.66 ±0.00 0.34^(#) 2.91^(##) 3.40^(##) 0.24^(##) 0.58^(#) Test drug 13 1.000.00 32.00 32.00 4.75 5.75 (100 mg/Kg) 14 1.00 1.00 23.00 23.00 4.434.73 15 0.00 2.00 32   32   7.25 7.85 16 1.00 1.00 22.00 26.00 6.22 6.9217 0.00 1.00 33.00 36.00 6.88 6.98 18 0.00 1.00 32.00 31.00 7.98 7.88Mean ± SEM 0.50 ± 1.00 ± 29.00 ± 30.00 ± 6.25 ± 6.69 ± 0.22 0.26 2.071.91 0.58* 0.50* Test drug 19 0.00 1.00 36.00 42.00 6 6.4 (200 mg/Kg) 201.00 1.00 25.00 34.00 7.69 7.09 21 0.00 1.00 27.00 32.00 6.09 6.9 221.00 1.00 29.00 27.00 6.88 6.08 23 1.00 0.00 33.00 24.00 6.67 6.77 241.00 0.00 38.00 37.00 5.44 5.04 Mean ± SEM 0.67 ± 0.67 ± 31.33 ± 32.67 ±6.46 ± 6.38 ± 0.21 0.21* 2.11* 2.68 0.32 0.31 The results are expressedin Individual as well as Mean ± SEM (n = 6); Statistical analysis wasdone using graph pad prism 5.0 version and Tukey post hoc test wasperformed. ^(#)p < 0.05, ^(##)p < 0.01 compared with normal control: p <0.05 (*) & 0.01 (**) compared with Gentamicin (100 mg/kg) i.p

TABLE 8 Effect of Test drug Treatment on Organ Weight in NephrotoxicityInduced Rats Treatment Animal No Kidney weight (g) Normal control 1 2.002 2.30 3 2.30 4 2.30 5 1.40 6 2.20 Mean ± SEM 2.08 ± 0.14 Gentamicin(100 mg/kg i.p) 7 1.90 8 2.50 9 2.70 10 2.30 11 2.70 12 2.90 Mean ± SEM2.50 ± 0.15 Test drug (100 mg/kg) 13 3.30 14 2.50 15 2.40 16 3.00 173.10 18 2.40 Mean ± SEM 2.78 ± 0.16 Test drug (200 mg/kg) 19 2.10 202.80 21 2.60 22 2.70 23 2.80 24 2.40 Mean ± SEM 2.57 ± 0.11

TABLE 9 Effect of Test drug Treatment on Blood Biochemistry InNephrotoxicity Induced Rats Animal ACP Albumin ALP Total protein γ-GTTreatment No U/L (g/dL) (U/L) (g/dl) (U/L) 0.5% CMC 1 38.00 2.57 59.005.67 1.00 2 33.00 2.49 55.00 5.40 1.00 3 38.00 2.51 55.00 5.53 1.00 432.00 2.45 57.00 5.34 1.00 5 50.00 2.71 69.00 6.32 1.00 6 31.00 2.4665.00 5.30 1.00 Mean ± SEM 37.00 ± 2.53 ± 60.00 ± 5.59 ± 1.00 ± 2.880.04 2.35 0.16 0.00 Gentamicin 7 42.00 2.58 83.00 6.25 2.00 (100 mg/kg)8 30.00 2.44 92.00 5.27 4.00 i.p 9 40.00 2.41 92.00 5.66 1.00 10 37.002.50 83.00 5.87 3.00 11 40.00 2.63 69.00 5.94 2.00 12 46.00 2.53 67.005.45 3.00 Mean ± SEM 39.17 ± 2.52 ± 81.00 ± 5.74 ± 2.50 ± 2.20 0.034.43^(##) 0.15 0.43^(##) Test drug 13 45.00 2.38 67.00 5.52 1.00 (100mg/Kg) 14 34.00 2.28 84.00 6.06 1.00 15 43.00 2.61 94.00 5.72 1.00 1633.00 2.55 62.00 5.37 2.00 17 30.00 2.51 69.00 5.50 1.00 18 45.00 2.5989.00 5.56 1.00 Mean ± SEM 38.33 ± 2.49 ± 77.50 ± 5.62 ± 1.17 ± 2.750.05 5.38 0.10 0.17** Test drug 19 41.00 2.66 70.00 5.72 1.00 (200mg/Kg) 20 36.00 2.71 76.00 6.41 1.00 21 52.00 2.98 87.00 6.91 1.00 2242.00 2.97 91.00 6.39 1.00 23 45.00 2.47 96.00 5.80 1.00 24 35.00 2.7397.00 5.62 1.00 Mean ± SEM 41.83 ± 2.75 ± 86.17 ± 6.14 ± 1.00 ± 2.55.080 4.48 0.21 0.00** Animal Creatinine Urea Uric acid GlobulinTreatment No (mg/dl) (mg/dl) (mg/dl) (g/dl) 0.5% CMC 1 0.64 33.00 1.153.10 2 0.64 42.00 1.85 2.91 3 0.63 29.00 1.42 3.02 4 0.88 31.00 1.212.88 5 0.74 34.00 1.83 3.61 6 0.69 35.00 1.75 2.84 Mean ± SEM 0.70 ±34.00 ± 1.54 ± 3.06 ± 0.04 1.83 0.13 0.12 Gentamicin 7 0.99 44.00 1.433.67 (100 mg/kg) 8 0.84 39.00 1.84 2.83 i.p 9 1.00 67.00 2.48 3.25 100.93 60.00 2.02 3.37 11 0.87 46.00 1.46 3.31 12 0.80 45.00 2.35 2.91Mean ± SEM 0.91 ± 50.17 ± 1.93 ± 3.22 ± 0.03^(##) 4.42^(##) 0.18^(##)0.13 Test drug 13 0.90 41.00 2.30 3.13 (100 mg/Kg) 14 1.02 52.00 2.573.79 15 0.81 43.00 1.03 3.11 16 0.71 27.00 1.45 2.82 17 0.88 40.00 1.293.00 18 0.76 37.00 1.60 2.96 Mean ± SEM 0.85 ± 40.00 ± 1.71 ± 3.13 ±0.05 3.33 0.25 0.14 Test drug 19 0.77 44.00 1.63 3.06 (200 mg/Kg) 200.65 35.00 0.84 3.70 21 1.10 43.00 1.18 3.93 22 0.88 43.00 1.08 3.42 230.87 42.00 1.24 3.33 24 0.71 39.00 1.74 2.89 Mean ± SEM 0.83 ± 41.00 ±1.29 ± 3.39 ± 0.07 1.39 0.14** 0.16 The results are expressed inIndividual as well as Mean ± SEM (n = 6); Statistical analysis was doneusing graph pad prism 5.0 version and Tukey post hoc test was performed.# p < 0.05, ^(##)p < 0.01 compared with Normal control: p < 0.05 (*) &0.01 (**) compared with Gentamicin (100 mg/kg) i.p

TABLE 10 Effect of Test drug Treatment on Serum Electrolytes inNephrotoxicity Induced Rats Animal K Na Cl nCa tCa Treatment No mmol/Lmmol/L mmol/L mmol/L mmol/L pH 0.5% CMC 1 12.27 144.61 101.56 3.3 6.657.91 2 12.67 146.68 102.34 1.7 3.4 7.94 3 14.01 147.27 105.49 2.25 4.557.95 4 11.8.3 147.27 107.10 2.5 5 7.92 5 11.89 145.79 103.11 2.7 4.857.89 6 12.99 147.87 106.70 0.65 1.35 7.91 Mean ± SEM 12.61 ± 146.58 ±104.38 ± 2.18 ± 4.30 ± 7.92 ± 0.33 0.49 0.96 0.37 0.73 0.01 Gentamicin 712.39 146.38 102.34 5.75 11.50 7.92 (100 mg/kg) 8 11.02 147.87 103.11 47.95 7.91 i.p 9 11.04 147.27 97.80 5.9 11.85 7.94 10 12.16 146.98 103.112.55 5.10 7.88 11 13.12 145.79 99.66 4.3 8.65 7.92 12 12.04 148.47100.42 3.5 7.00 7.98 Mean ± SEM 11.96 ± 147.13 ± 101.07 ± 4.33 ± 8.68 ±7.93 ± 0.33 0.40 0.88 0.53^(##) 1.07^(##) 0.01 Test drug 13 12.72 149.78106.77 2.8 5.65 7.89 (100 mg/Kg) 14 11.36 146.63 101.04 3.05 6.05 7.9015 12.37 158.40 113.82 3.5 7.00 7.89 16 12.45 145.34 105.21 2.7 6.757.92 17 17.38 147.20 112.37 4.05 8.10 7.79 18 13.86 153.58 99.77 2.254.50 7.93 Mean ± SEM 13.36 ± 150.16 ± 106.50 ± 3.06 ± 6.34 ± 7.89 ± 0.872.03 2.34 0.26 0.50 0.02 Test drug 19 10.16 154.18 99.77 1.65 3.30 7.91(200 mg/Kg) 20 17.69 145.79 93.27 3.1 6.20 7.99 21 14.18 146.91 98.930.3 0.60 8.11 22 13.48 141.36 92.50 1.5 2.95 8.08 23 11.56 140.27 90.961.9 3.85 8.00 24 13.48 145.68 95.14 4.4 8.80 8.04 Mean ± SEM 13.43 ±145.70 ± 95.10 ± 2.14 ± 4.28 ± 8.02 ± 1.05 2.01 1.46 0.58** 1.16** 0.03The results are expressed in Individual as well as Mean ± SEM (n = 6);Statistical analysis was done using graph pad prism 5.0 version andTukey post hoc test was performed. # p < 0.05, ^(##)p < 0.01 comparedwith Normal control: p < 0.05 (*) & 0.01 (**) compared with Gentamicin(100 mg/kg) i.p

TABLE 11 Effect of Test drug Treatment on Renal Stress Marker InNephrotoxicity Induced Rats GPx Animal GSH (mg/mt/mg TBARS SOD CatalaseTreatment No (mg/g) ptn) (mg/g) U/mt/g ptn mm/mt/mg ptn 05% CMC 1 1.0613.72 0.73 19.02 146.54 2 1.53 14.80 0.69 10.84 326.90 3 1.65 13.26 0.6117.75 189.92 4 1.97 11.13 0.43 32.56 433.56 5 2.99 20.49 0.48 36.73417.93 6 2.64 19.27 0.52 31.92 414.95 Mean ± SEM 1.97 ± 15.44 ± 0.58 ±24.80 ± 338.63 ± 0.29 1.49 0.05 4.21 41.58 Gentamicin 7 0.83 5.79 0.79 9.02 179.44 (100 mg/kg) 8 1.90 7.12 0.90 10.34 225.04 i.p 9 0.16 6.820.93 14.22 280.37 10 1.78 11.42 0.73 12.90 287.86 11 0.94 7.24 0.8722.26 339.18 12 0.17 3.06 0.88 15.12 127.34 Mean ± SEM 0.96 ± 6.91 ±0.85 ± 13.97 ± 239.87 ± 0.31^(##) 1.10^(##) 0.03^(#) 1.91^(##) 31.80Test drug 13 1.38 10.69 0.60 18.10 254.22 (100 mg/Kg) 14 2.36 8.72 0.8214.96 206.63 15 2.28 10.53 0.53 18.08 272.51 16 1.16 5.83 0.55 10.02254.38 17 1.58 4.60 0.52 17.91 205.98 18 0.91 5.74 0.74  8.68 257.08Mean ± SEM 1.61 ± 7.68 ± 0.63 ± 14.63 ± 241.80 ± 0.24** 1.08 0.05* 1.7511.56 Test drug 19 2.85 8.79 1.07 13.73 379.07 (200 mg/Kg) 20 2.35 7.580.95 15.16 242.37 21 2.06 7.66 0.47 19.03 245.98 22 1.72 10.06 0.5119.34 259.08 23 2.02 6.07 0.59 11.98 177.58 24 1.91 8.06 1.05 15.06271.82 Mean ± SEM 2.15 ± 8.04 ± 0.77 ± 15.72 ± 262.65 ± 0.16** 0.54 0.111.19 26.82 The results are expressed in Individual as well as Mean ± SEM(n = 6); Statistical analysis was done using graph pad prism 5.0 versionand Tukey post hoc test was performed. ^(#)p < 0.05, ^(##)p < 0.01compared with Normal control: p < 0.05 (*) & 0.01 (**) compared withGentamicin (100 mg/kg) i.p

TABLE 12 Individual Animal Histopathological Findings Animal MicroscopicFindings Group No. Kidney Liver I 1 Tubular dilatation, cortex, minimalInflammatory cells, focal, minimal Tubular degeneration & Congestedportal, central vein, regeneration, minimal and sinusoids agonal changesInflammatory cells infiltration, BRL Increase cytoplasmic glycogenHemorrhages, agonal changes contents 2 Tubular dilatation, cortex,minimal Cytoplasmic vacuolation, Tubular degeneration & hepatocytes,minimal regeneration, minimal Increase cytoplasmic glycogen Congestion,agonal changes contents Hemorrhages, agonal changes Congested portal,central vein, and sinusoids, agonal changes 3 Tubular dilatation,cortex, minimal Inflammatory cells, multifocal, Tubular degeneration &minimal regeneration, minimal Increase cytoplasmic glycogen Inflammatorycells infiltration, contents minimal Congested portal, central vein, andCongestion, agonal changes sinusoids, agonal changes Hemorrhages, agonalchange 4 Tubular dilatation, cortex, minimal Increase cytoplasmicglycogen Tubular degeneration & contents regeneration, minimalInflammatory cells infiltration, minimal Congestion, agonal changesHemorrhages, agonal changes 5 and 6 Tubular dilatation, cortex, minimalIncrease cytoplasmic glycogen Tubular degeneration & contentsregeneration, minimal Congested portal, central vein, and Congestion,agonal changes sinusoids, agonal changes Hemorrhages, agonal changes II7 Tubular dilatation, cortex, moderate Increase cytoplasmic glycogenInflammatory cells infiltration, contents multifocal, moderateInflammatory cells infiltration, Tubular degeneration & minimalregeneration with dilatation, Sinusoidal congestion, agonal multifocal,moderate change Tubular cell necrosis, minimal Tubular cell vacuolation,minimal proteineous fluid, minimal, Hemorrhages, agonal changes,Congestion, agonal changes II 8 Tubular dilatation, cortex, moderateIncrease cytoplasmic glycogen Inflammatory cells infiltration, contentsmultifocal, moderate Tubular Inflammatory cells infiltration,degeneration & regeneration with minimal dilatation, multifocal,moderate Single cell necrosis, minimal Tubular cell vacuolation, minimalProteineous fluid, minimal Congestion, agonal changes 9 Tubulardilatation, cortex, moderate Increase cytoplasmic glycogen Inflammatorycells infiltration, contents multifocal, moderate Tubular degeneration &regeneration with/without dilatation, multifocal, moderate Tubular cellvacuolation, minimal proteineous fluid, minimal Congestion, agonalchanges 10 Tubular dilatation, cortex, moderate Inflammatory cellsinfiltration, Inflammatory cells infiltration, peribilliary mildmultifocal, mild Tubular degeneration & regeneration with/withoutdilatation, multifocal, moderate Tubular cell vacuolation, minimalproteineous fluid, minimal Congestion, agonal changes 11 Tubulardilatation, cortex, moderate Increase cytoplasmic glycogen Inflammatorycells infiltration, mild contents moderate Tubular degeneration &regeneration Sinusoidal congestion, agonal with/without dilatation,multifocal, changes moderate Tubular cell vacuolation, minimal Tubularcell necrosis, minimal II 12 Tubular dilatation, cortex, Inflammatorycells infiltration, marked minimal Inflammatory cells infiltration,minimal Tubular degeneration & regeneration with/without dilatation,multifocal, moderate Tubular cell vacuolation, minimal Tubular cellnecrosis, minimal Inflammatory cells infiltration, glomeruli, minimalIII 13 Tubular dilatation, cortex, mild Sinusoidal congestion, agonalInflammatory cells infiltration, changes multifocal, mild Tubulardegeneration & regeneration with/without dilatation, multifocal, mildCongestion, agonal changes Hemorrhages, agonal changes 14 Tubulardilatation, cortex, mild Inflammatory cells infiltration, Inflammatorycells infiltration, minimal multifocal, mild Tubular Increasecytoplasmic glycogen degeneration & regeneration contents with/withoutdilatation, multifocal, mild Congestion, agonal changes Hemorrhages,agonal changes 15 Tubular dilatation, cortex, NAD moderate Inflammatorycells infiltration, multifocal, moderate Tubular degeneration &regeneration with/without dilatation, multifocal, moderate Tubular cellvacuolation, minimal Hemorrhages, agonal changes III 16 Tubulardilatation, cortex, Inflammatory cells infiltration, moderate minimalInflammatory cells infiltration, multifocal, moderate Tubulardegeneration & regeneration with/without dilatation, multifocal,moderate Tubular cell vacuolation, minimal 17 Tubular dilatation,cortex, Cytoplasmic vacuolation, moderate hepatocytes, mild SinusoidalInflammatory cells infiltration, congestion, agonal changes multifocal,moderate Tubular degeneration & regeneration with/without dilatation,multifocal, moderate Tubular cell vacuolation, minimal, Congestion,agonal changes Hemorrhages, agonal changes 18 Tubular dilatation,cortex, mild Cytoplasmic vacuolation, Inflammatory cells infiltration,hepatocytes, minimal multifocal, mild Tubular degeneration &regeneration with/without dilatation, multifocal, mild Congestion,agonal changes IV 19 Tubular dilatation, cortex, Inflammatory cellsinfiltration, moderate minimal Inflammatory cells infiltration, Increasecytoplasmic glycogen multifocal, moderate Tubular contents degeneration& regeneration Sinusoidal Congestion, agonal with/without dilatation,changes multifocal, moderate Tubular cell vacuolation, minimal IV 20Tubular dilatation, cortex, mild Cytoplasmic vacuolation, Inflammatorycells infiltration, hepatocytes, minimal Sinusoidal multifocal, mildTubular Congestion, agonal changes degeneration & regenerationwith/without dilatation, multifocal, mild Tubular cell vacuolation,minimal Congestion, agonal changes 21 Tubular dilatation, cortex, NADminimal Inflammatory cells infiltration, multifocal, mild Tubulardegeneration & regeneration with/without dilatation, multifocal, minimalCongestion, agonal changes 22 Tubular dilatation, cortex, Cytoplasmicvacuolation, minimal hepatocytes, minimal Sinusoidal Inflammatory cellsinfiltration, Congestion, agonal changes multifocal, mild Tubulardegeneration & regeneration with/without dilatation, multifocal, minimalCongestion, agonal changes IV 23 Tubular dilatation, cortex, mild NADInflammatory cells infiltration, multifocal, mild Tubular degeneration &regeneration with/without dilatation, multifocal, mild Tubular cellvacuolation, minimal Congestion, agonal changes 24 Tubular dilatation,cortex, mild Cytoplasmic vacuolation, Inflammatory cells infiltration,hepatocytes, minimal Increase multifocal, mild Tubular cytoplasmicglycogen contents degeneration & regeneration with/without dilatation,multifocal, mild Hemorrhages, agonal changes Congestion, agonal changes

TABLE 13 Summary of Grading System for Histopathology Evaluation ofKidney Group/Treatment/No. of animals III/ IV- Test drug- Test drug- I/II/ low dose High dose Normal Positive (100 mg/ (200 mg/ Lesionscontrol/6 control/6 kg)/6 kg)/6 1. Tubular dilatation, 6 5 6 6 cortexMinimal 6 0 0 2 Mild 0 0 3 3 Moderate 0 4 3 1 Marked 0 1 0 0 2. Tubulardegeneration 6 6 6 6 and regeneration Minimal 6 0 0 2 Mild 0 0 3 3Moderate 0 6 3 1 Marked 0 0 0 0 3. Inflammatory cells 2 6 6 6infiltration, multifocal Minimal 2 1 0 0 Mild 0 2 3 5 Moderate 0 3 3 14. Inflammatory cells 1 0 0 0 infiltration, glomeruli Minimal 1 0 0 0 5.Tubular cell 0 6 3 3 vacuolation Minimal 0 6 3 3 Mild 0 0 0 0 Moderate 00 0 0 Marked 0 0 0 0 6. Proteineous fluid 0 4 0 0 Minimal 0 4 0 0 7.Tubular cell necrosis 0 4 0 0 Minimal 0 4 0 0 Mild 0 0 0 0 Marked 0 0 00

TABLE 14 Summary of Grading System for Histopathology Evaluation ofLiver Group/Treatment/No. of animals III/ IV- Test drug- Test drug- I/II/ low dose High dose Normal Positive (100 mg/ (200 mg/ Lesionscontrol/6 control/6 kg)/6 kg)/6 1. Inflammatory cells 2 3 2 1infiltration Minimal 2 3 2 1 Mild 0 0 0 0 2. Hepatocytes 1 0 1 2cytoplasmic vacuolation Minimal 1 0 0 2 Mild 0 0 1 0 3. Single cellnecrosis 0 1 0 0 Minimal 0 1 0 0 4. Inflammatory cell 0 1 0 0infiltration, peribilliary Mild 0 1 0 0 5. Increase cytoplasmic 4 4 0 1glycogen contentsConclusion:

The results thereby demonstrate that Test drug has potentnephroprotective action upon Gentamycin-induced renal damage in rats andpossess antilipid peroxidative and free radical scavenging activities.Further, it demonstrates that treatment with Test drug at 200 mg/kg inrats can prevent functional as well as histological renal changesinduced by gentamicin.

Example 2: Clinical Study

Aims and objectives: To prove the efficacy of the Test drug inmanagement of chronic renal failure (CRF). To give the scientificestablishment to the observations which show that Test drugsignificantly correct albuminuria and serum creatinine value, which arethe cardinal features of CRF, and improve the renal function which isevident by reduction in serum creatinine and blood urea levels.

Materials and Methods: All the patients were selected from the OPD andIPD of the Muniyal Ayurveda Hospital and Research Centre Manipal.

Criteria for inclusion: Patients with clinically positive history ofCRF, having the clinical features of CRF like albuminuria, raised serumcreatinine and blood urea, were included.

Criteria for exclusion: The patients having diabetic nephropathy wereexcluded from this study. Patients who were on dialysis therapy.Patients with other added complications.

Plan of study and management: A total of 100 patients with CRF wereregistered for the study. The patients were administered with the Testdrug tablet, 2 tablets (2×500 mg) twice a day. The patients were kept onnormal healthy diet. The doses of the continuous antihypertensive drugsof the patients were not interfered with. Duration of the treatment was1 month.

Assessment of results: All the patients were clinically assessed beforeand after treatment. Changes in symptoms, albuminuria, serum creatinine,blood urea and hemoglobin were observed. Observations were evaluatedstatistically.

Results and Observations: The main causative factor for CRF in thisstudy was hypertension; 86% patients were found in this group. 7%patients were having chronic nephritis and 5% were having polycystickidney disease. Only 2% of the patients were found with obstructivenephropathy. Serum creatinine reduced by 20.71% and it was statisticallyhighly significant.

Blood urea reduced by 32.15% and it was also statistically highlysignificant. Albuminuria reduced by 36.70% and this was statisticallyhighly significant. Hemoglobin increased by 4.65% and this wasstatistically highly significant. None of the patients had shown any newand unusual feature. Urine output increased by 56.54% and it wasstatistically highly significant. Out of 100 patients, edema wasreported by 58 patients and it reduced by 71.56%. Nausea was reported in46 patients and it was relieved by 68.75%. The 24 patients were found tohave vomiting and it was relieved by 82.35%. Weakness was found in 85patients and it reduced by 55.61%. The 53 patients were having loss ofappetite, and appetite increased by 59.29%. Leg cramps were found in 17patients and it reduced by 72%. Breathlessness was found in 27 patientsand it was relieved by 64.29%. Hiccup was found in only one patient andwas totally relieved. The 19 patients reported pruritus and it reducedby 66.66%. Reductions in all these symptoms were statistically highlysignificant.

Discussion and Interpretation: As mentioned earlier, CRF is specificform of renal disease. According to Ayurveda, CRF is a disease ofMutravaha Srotas. Though all the three Doshas as well as all the Dushyasare involved in the disease, Kapha is responsible in blockingmicrovessels and developing microangiopathy. Vata is responsible fordegeneration of the structure of the kidney. According to Ayurvedicprinciples of management of the disease, tissue damage can be preventedand repaired by Rasayana drugs because they have the capability toimprove qualities of tissues and hence increase resistance of thetissues. On the other hand, blockage can be removed by Lekhana drugshaving scraping effect on blocked channels.

In this study, it was found that serum creatinine reduced by 20.71% withtreatment. This beneficial effect was statistically highly significantwhich is encouraging and shows improvement in kidney functions. Bloodurea reduced by 36.15% with treatment and it was also statisticallyhighly significant. Treatment resulted in reduction in albuminuria by36.70% and increase in hemoglobin by 4.65%, which were statisticallyhighly significant. Reduction in edema, weakness, leg cramps, increasein appetite, and relief from nausea, breathlessness and pruritus werestatistically highly significant.

Conclusion: With the help of clinical observations and the discussionmade, it may be concluded that 86% patients of CRF have hypertension asa basic underlying cause. The result obtained may be attributed to thedisease modifying effect of trial therapy by means of its Rasayana andanti Vata-Kapha properties. The trial therapy is an ideal drug as a safeand effective alternative in case of CRF.

Serum creatinine, blood urea and albuminuria reduced 20.71%, 32.15% and36.70%, respectively. Hemoglobin level and urine output increased by4.38% and 56.54%, respectively. They were statistically highlysignificant. All the patients had shown more than 50% relief in all thesigns and symptoms.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the embodiments herein that others can, byapplying current knowledge, readily modify and/or adapt for variousapplications such specific embodiments without departing from thegeneric concept, and, therefore, such adaptations and modificationsshould and are intended to be comprehended within the meaning and rangeof equivalents of the disclosed embodiments. It is to be understood thatthe phraseology or terminology employed herein is for the purpose ofdescription and not of limitation. Therefore, while the embodimentsherein have been described in terms of preferred embodiments, thoseskilled in the art will recognize that the embodiments herein can bepracticed with modification within the spirit and scope of theembodiments as described herein.

I claim:
 1. A process for preparation of a formulation for treatment andmanagement of Renal disorders, comprising: levigating bhasma, Gugguluand shilajit, wherein said bhasma is at least one selected from thegroup consisting of Swarna Makshika bhasma, Mandura bhasma and Lohabhasma; adding herbs, alkali and salt, wherein said herbs compriseCinnamomum camphora, Acorus calamus, Saussurea lappa, Andrographispaniculata, Tinospora cordifolia, Cedrus deodara, Curcuma longa,Aconitum heterophyllum, Berberis aristata, Plumbago rosea, Coriandrumsativum, Emblica officinalis, Terminalia chebula, Terminalia bellerica,Piper chaba, Embelia ribes, Piper longum, Piper nigrum, Commiphoramukul, Saccharum officinarum, Zingiber officinalis, Operculinaturpethum, Baliospermum montanum, Cinnamomum tamala, Cinnamomumzeylanica, Elettaria cardamomum, Bamboo manna, Boerhavia diffusa,Cyperus rotundas, Bauhinia variegata and Holarrhena antidysenterica; andadding grinding decoction, fresh juice and gomutra while continuinggrinding to obtain a ground mass, wherein said grinding decoctioncomprises decoction of Cyperus rotundas, Piper longum, Asparagusracemosus, Bauhinia variegata, Oldenlandia umbellata, Hemidesmusindicus, Vetiveria zizanioides, Emblica officinalis, Terminalia chebula,Terminalia bellerica and Boerhavia diffusa, and wherein said fresh juicecomprises juice of Aloe vera, Punica granatum, Cynodon dactylon andAnanas comosus.
 2. The process as claimed in claim 1, wherein said herbscomprise finely powdered form of dried root of Acorus calamus, Saussurealappa, Aconitum heterophyllum, Berberis aristata, Piper longum, Piperchaba, Plumbago rosea, Operculina turpethum, Baliospermum montanum,Elettaria cardamomum, Boerhavia diffusa and Cyperus rotundas; finelypowdered dried fruits of Coriandrum sativum, Emblica officinalis,Terminalia chebula, Terminalia bellerica, Piper longum, Embelia ribes,and Piper nigrum; crystals of Cinnamomum camphora; finely powdered driedheartwood of Cedrus deodara; finely powdered dried leaves of Cinnamomumtamala; finely powdered dried whole plant of Andrographis paniculata;finely powdered dried rhizome of Zingiber officinalis and Curcuma longa;finely powdered dried stem of Tinospora cordifolia, Saccharumofficinarum and Piper chaba; finely powdered dried stem bark ofCinnamomum zeylanica, Bauhinia variegata and Holarrhena antidysenterica;secretion of Bamboo manna; and oleo-gum resin of Commiphora mukul. 3.The process as claimed in claim 1, wherein said alkali comprises atleast one alkali selected from a group consisting of Yavakshara andSarjakshara.
 4. The process as claimed in claim 1, wherein said saltcomprises at least one salt selected from a group consisting of Rocksalt, Sonchal salt and Black salt.
 5. The process as claimed in claim 1,wherein said grinding decoction is a decoction of dry root of Cyperusrotundas, dried fruit of Piper longum, dried root of Asparagusracemosus, dried stem bark of Bauhinia variegata, dried whole plant ofOldenlandia umbellata, dry root of Hemidesmus indicus, dry root ofVetiveria zizanioides, dry fruit of Emblica officinalis, dry fruit ofTerminalia chebula, dry fruit of Terminalia bellerica and dry root ofBoerhavia diffusa.